This study used lysozyme as a model protein to investigate protein structure dynamics in aqueous solutions under accelerated stress conditions using various techniques including Raman spectroscopy, circular dichroism, size exclusion chromatography, and particle analysis. Lysozyme samples formulated at 50mg/ml were incubated at 40°C and 65°C for up to one week. Raman spectroscopy detected perturbations in protein structure like disulfide bonds and amino acids in samples incubated at 65°C after 4 days but not at 40°C. Size exclusion chromatography and particle analysis also showed increased aggregation and particles at 65°C but not 40°C. The results suggest Raman spectroscopy can reliably predict protein structure integrity during stress testing.

1. Introduction
Materials and Methods
Results
Spectroscopic Characterization of Protein Structure Dynamics in Aqueous States
Yemin Xu1, Chris Carpenter2, Korben Knudson3, Rina Dukor4, John Carpenter5, Theodore Randolph3
1Biochemistry Department, University of Colorado Boulder
2Dept. of Physics, University of Colorado Boulder
3Dept. of Chemical and Biological Engineering, University of Colorado Boulder
4BioTools, Inc
5Dept. of Pharmaceutical Sciences, University of Colorado, Denver
Maintaining protein high order structure integrity is critical for
developing protein pharmaceuticals. Mis-folded or unfolded protein
pharmaceuticals will compromise their efficacy, change the
pharmacokinetics profile and/or cause severe immune-responses.
Thus, it is important to develop fast and accurate methods to
routinely monitor the proteins pharmaceutical’s structures.
In this study, lysozyme was used as model protein, formulated at
50mg/ml and incubated at elevated temperatures (40 and 65 °C) for
one week. Raman spectroscopy and Circular Dichroism (CD) were
employed to measure the lysozyme structure in aqueous solution.
Particle formation was monitored by Flowcam, and soluble
aggregates were examined and quantified by size exclusion
chromatography (SEC).
The extent of structural perturbation observed during incubation
exhibited good correlation with the rate of aggregation and sub-
visible particle formation under accelerated conditions. The results
demonstrate that Raman spectroscopy could be a quick and reliable
way to predict protein stability and high order structure integrity
during incubation.
50mg/mL
Lysozyme (H2O)
pH 4.0
Samples divided
into two groups
(40oCand 65oC)
40oC 65oC
Samples were placed in the incubators for measuring at various
time points (0-Point, Day 1, Day 4 , and Day 7, respectively).
At each time point, triplicate samples were removed from the
incubator and measured by different methods.
Lysozyme from chicken egg white (Sigma-L6876):
50mg/ml (H2O, pH 4.0)
Incubation temperature:
40±2 and 65±2oC
Incubation time point:
0, 1, 4 and 7 days
Protein particle measurement:
Flowcam (0.2mL sample size)
Soluble protein aggregation:
SEC (mobile phase: 0.1 M Phosphate buffer 0.1 M Na2SO4, pH 6.7 )
Non-Reducing SDS Gel
Proteins structure measurement:
CD (Far-UV 0.3mg/ml, Near-UV 50mg/ml)
Raman (50mg/ml, laser power 100mW at 780nm)
2013 Colorado Protein Stability Conference
Discussion
Fig 4: Protein secondary and tertiary structure measured by circular dichroism (CD)
A) Far-UV: measuring secondary structure; B) near-UV: measuring tertiary structure.
Each color curve is the average of triplicate result. Different colors show different samples incubated at different temperatures.
CD data show similar secondary structures for all samples, however, tertiary structures measured by near-UV indicate protein
tertiary strcutures of samples incubated at 65oC differ from others after 4 days. Each spectra represents average of triplicate
measurements.
A B
A
Disulfide bond S-S
C
Amide III
B
Tyrosine
D
Amide I
Fig 1: Protein structure measured by Raman
Center panel is the entire spectra of 50mg/ml lysozyme in aqueous solution (control) and 80oC for 20 hours.
Close look (A, B, C, D)of each representative region of disulfide bond, tyrosine, amide III and amide I indicates that protein
structures were significantly perturbed after storing at high temperature (80oC).
A
Disulfide bond S-S
C
Amide III
B
Tyrosine
D
Amide I
Fig 2: Raman spectra of proteins incubated for various times at 40oC
Center panel is the entire spectra of 50mg/ml lysozyme at 40oC for different time points
Close look (A, B, C, D)of each representative region of disulfide bond, tyrosine, amide III and amide I indicates that protein
structures were similar after storing at 40oC for 1 week. Each spectra represents average of triplicate measurements.
A
Disulfide bond S-S
C
Amide III
B
Tyrosine
D
Amide I
Fig 3: Raman spectra of proteins incubated for various times at 60oC
Center panel is the entire spectra of 50mg/ml lysozyme at 60oC for different time points
Close look (A, B, C, D)of each representative region of disulfide bond, tyrosine, amide III and amide I indicates that protein
structures were disrupted after 4 days storing at 60oC. Each spectra represents average of triplicate measurements.
Fig 5: percentage of remaining monomer measured by SEC
Results of triplicate measurements show that no
noticeable protein loss was seen for samples incubated at
40oC for one week, in contrast, there was around 10%
monomer lysozyme lost after one week at 65oC.
Fig 6: non-reducing SDS gel (coomassie blue stain)
Non-reducing SDS gel shows that there are noticeable
aggregates from samples incubated at 65oC after 4 days.
MW kD
50
37
25
20
15
10
Fig 7: Sub-visible particle concentration measured by flowcam
Within the flowcam measurable size range, protein particle size and
concentration increase for both 40oC and 60oC samples. Representative
images from each size range are shown
Conclusions References
 Structure information from Raman spectroscopy and CD measurement show that there are structure perturbations in
samples incubated at 65oC after 4 days.
 Particle concentration decreased after 4 days, it may due to the size of protein aggregates is beyond the capable range of
flowcam
 Far-UV measurement did not pick up noticeable differences between samples, but near-UV did, which indicates that
under experiment condition, lysozyme likely undergoes tertiary structural perturbation.
 Raman spectroscopy is a useful tool to examine the protein high order structure, and it is capable to identify the
structural differences occurring at various position (eg. Disulfide bond, tyrosine, amide III and amide I region).)
 It is critical and helpful to use orthogonal methodologies to evaluate protein stability.
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